Mass spectrometry has been used to determine the extent of modification and the specific sites of modification on biomolecules. MS-based approaches have many advantages, including generally rapid analyses without radiolabeling. The MS analysis of a variety of proteins has been investigated using mass spectrometry. Products and digests have been analyzed by both positive and negative ion MALDI mass spectrometry and LC in combination with electrospray mass spectrometry. In addition, we are currently analyzing the use of the crosslinker BS3 with a variety of proteins. 1. Antibody Glycosylation Characterization. Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are considered pathogenic in ANCA glomerulonephritis and vasculitis (AAV). ANCAs are predominantly of the IgG isotype. IgG molecules contain an N-glycan on each of their Fc CH2 domains. In addition, 15-25%of serum IgG contains glycans within the Fab variable domain. Modification of the serum IgG glycoform profile, particularly in the Fc region, has been reported as a factor in the pathogenesis in several inflammatory autoimmune diseases including ANCA disease. However, the respective role of IgG Fc and Fab glycosylation in ANCA pathogenicity is largely unexplored. We showed significant differences between patients with MPO- and PR3-ANCA diseases with respect to changes with disease activity in the amount and type of glycans on the IgG Fc portion. The prevalence of Fab glycosylation sites on anti-MPO specific IgG is significantly increased compared to non-autoreactive IgGs. 2. Histone Proteins. We have been collaborating with the Archer laboratory in an effort to examine the role of H1 in controlling gene expression and protein levels when we knock out the H1 gene. Knockout cell lines of the histone protein H1.4 protein (as it was determined by MS previously that it was phosphorylated) was compared to a wild-type cell line. Data were acquired and are being analyzed to determine if other histone levels change and/or their PTMS change if the H1.4 isoform is knocked out. 3. Protein Crosslinking. Multiple projects are underway to characterize protein complexes by mass spectrometry in conjunction with chemical cross-linking. Most of these experiments have been conducted using BS3 as the cross-linking reagent followed by trypsin digestion and nanoLC-ESI-MS performed on a Q-Exactive Plus mass spectrometer. While there have been successful analyses on multiple projects, the most mature of these projects has been the characterization of the Grc3-Las1 complex. The newly discovered endoribonuclease (RNase) Las1 and the poly-nucleotide kinase (PNK) Grc3 assemble into the multienzyme complex. In an effort to understand how the Grc3-Las1 complex coordinates its dual enzymes, multiple structural approaches were used including chemical cross-linking and mass spectrometry as well as cryo-electron microscopy. The multiple structures were solved, and they reveal that the Grc3-Las1 complex adopts a butterfly-like architecture harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. 4. Phosphorylation of VRK1. Vaccinia-related kinase 1 (VRK1) is a Ser-Thr kinase and regulates numerous proteins. We found that VRK1 auto-phosphorylates at Ser376 and Thr386 in in vitro kinase assays. It was found that in cells, that Thr386 is phosphorylated in the low glucose (40 mg/dL) but not high glucose (140 mg/dL) media. This Thr386 phosphorylation correlates with an induced phosphorylation of p53 (Thr18) and C-Jun (Ser63) in the low glucose medium. Knock down of VRK1 attenuates phosphorylation of both C-Jun and p53 in the low glucose medium, while over-expression of VRK1 T386D (but not VRK1 WT or VRK1 T386A mutant) induces phosphorylation of p53 and C-Jun in the high glucose medium. 5. Peanut Proteomics. In collaboration with B. London and G. Mueller, we have previously investigated the AGE modifications of the major peanut allergens using mass spectrometry. The goal of the current work is to develop a proteomic screening of potential AGE biomarkers for understanding the effects of thermal processing on peanuts.